Every year, significant portions of the world's commercially important agricultural crops, including foods, textiles, and various domestic plants are lost to pest infestation, resulting in losses in the millions of dollars. Various strategies have been used in attempting to control such pests.
One strategy is the use of broad spectrum pesticides, chemical pesticides with a broad range of activity. However, there are a number of disadvantages to using such chemical pesticides. Specifically, because of their broad spectrum of activity, these pesticides may destroy non-target organisms such as beneficial insects and parasites of destructive pests. Additionally, these chemical pesticides are frequently toxic to animals and humans, and targeted pests frequently develop resistance when repeatedly exposed to such substances.
Another strategy has involved the use of biopesticides, which make use of naturally occurring pathogens to control insect, fungal and weed infestations of crops. Biopesticides are naturally occuring organisms that produce a toxin(s), a substance toxic to the infesting agent which is generally less harmful to non-target organisms and the environment as a whole than chemical pesticides.
The most widely used biopesticide is Bacillus thuringiensis (B.t.). B.t. is a widely distributed, rod shaped, aerobic and spore forming microorganism. During its sporulation cycle, B.t. produces a protein(s) known as a delta-endotoxin(s), that forms crystalline inclusion bodies within the cell. The delta-endotoxins have molecular weights ranging from 27-140 kD and kill insect larvae upon ingestion.
Delta-endotoxins have been produced by recombinant DNA methods (see, for example, Tailor et al., 1992, Molecular Microbiology 6:1211-1217; toxin is active against lepidopteran and coleopteran pests; Payne et al., U.S. Pat. No. 5,045,469; toxin is active against lepidopteran pests). The delta-endotoxins produced by recombinant DNA methods may or way not be in crystal form.
A number of B.t. strains have been isolated that have been found to be active against insect pests of the order Lepidoptera. B.t. subsp. kurstaki HD-1 produces bipyramidal and cuboidal crystal proteins in each cell during sporulation (Luthy et al., in Microbial and Viral Pesticides, ed. E. Kurstak, Marcel Dekker, New York, 1982, pp. 35-74); the bipyramidal crystal was found to be encoded by three cryIA genes (Aronson et al., 1986, Microbiol. Rev. 50:1-50). B.t. subsp. kurstaki HD-73 crystal delta-endotoxin contains the CryIA(c) protein (Adang et al., 1985, Gene 36:289-300). B.t. subsp. dendrolimus HD-7 and HD-37 contain a CryIA and a CryII protein; B.t. subsp. sotto contains an alkaline soluble protein that differs from the holotype CryIA(a) protein by 24 amino acids; B.t. subsp. subtoxicus HD-10 contains CryIA and CryIB proteins; B.t. subsp. tolworthi HD-121 contains CryIA and CryII proteins; and B.t. subsp. aizawai HD-68 contains CryIA proteins (Hofte and Whiteley, 1989, Microbiol. Reviews 53:242-255). Payne, U.S. Pat. No. 4,990,332, issued Feb. 5, 1993, discloses an isolate of B.t., PS85AI, and a mutant of the isolate, PS85AI, which both have activity against Plutella xylostella, a lepidopteran pest, and produce alkaline soluble proteins having a molecular weight of 130,000 and 60,000 daltons. Payne, U.S. Pat. No. 5,045,469, issued Sep. 3, 1991 discloses a B.t. isolate designated PS81F which also produces alkaline soluble proteins having a molecular weight of 130,000 and 60,000 daltons and has activity against Spodoptera exigua and T. ni; the toxin gene from PS81F appears to have little homology to the toxin gene from B.t. subsp. kurstaki HD-1. Payne, U.S. Pat. No. 5,206,166, filed Jun. 25, 1992, issued Apr. 27, 1993, discloses B.t. isolates PS81A2 and PS81RR1 which produce 133,601 and 133,367 dalton alkaline-soluble proteins; both have activity against Trichoplusia ni, Spodoptera exigua and Plutella xylostella and are different from B.t. Subsp. kurstaki HD-1 and other B.t. isolates Bernier et al., U.S. Pat. No. 5,061,489 and WO 90/03434 discloses strain A20 producing a delta-endotoxin encoded by at least three genes: 6.6-, 5.3-, and 4.5-type genes (cryIA(a), cryIA(b), and cryIA(c)). Chestukhina et al., 1988, FEBS Lett. 232:249-51, disclose that B.t. subsp. galleriae produces two delta-endotoxins, both of which are active against lepidopteran pests.
Other strains, e.g. Bacillus thuringiensis subsp. tenebrionis (Krieg et al., 1988, U.S. Pat. No. 4,766,203), have been found to be specific for Coleoptera. The isolation of another coleopteran toxic Bacillus thuringiensis strain was reported in 1986 (Hernnstadt et al. Bio/Technology vol. 4, 305-308, 1986, U.S. Pat. No. 4,764,372, 1988). This strain, designated "Bacillus thuringiensis subsp. san diego", M-7, has been deposited at the Northern Regional Research Laboratory, USA under accession number NRRL B-15939. However, the assignee of the '372 patent, Mycogen, Corp. has publicly acknowledged that Bacillus thuringiensis subsp. san diego is Bacillus thuringiensis subsp. tenebrionis.
Other isolated strains have been found to be active against two orders of pests. Padua, 1990, Microbiol. Lett. 66:257-262, discloses the isolation of two mutants containing two delta-endotoxins, a 144 kD protein having activity against a lepidopteran pest and a 66 kD protein having activity against mosquitoes. Bradfish et al., U.S. Pat. No. 5,208,017, discloses B.t. isolates PS86A1 and PS86Q3 which produce alkaline soluble proteins having a molecular weight of 58,000 and 45,000 daltons and 155,000, 135,000, 98,000, 62,000, and 58,000 daltons, respectively and which have activity against lepidopteran and coleopteran pests. PCT Application No. WO 90/13651 and Tailor et al., 1992, Molecular Microbiology 6:1211-1217, disclose a B.t. strain which is toxic against Lepidoptera and Coleoptera and which produces a toxin having a molecular weight of 81 kd.
It is advantageous to isolate new strains of Bacillus thuringiensis to produce new toxins so that there exists a wider spectrum of biopesticides for any given insect pest.